The present invention relates generally to the field of immunology and specifically to synthetic human monoclonal antibodies that bind and neutralize human immunodeficiency virus (HIV).
1. HIV Immunotherapy
HIV is the focus of intense studies as it is the causative agent for acquired immunodeficiency syndrome (AIDS). Immunotherapeutic methods are one of several approaches to prevention, cure or remediation of HIV infection and HIV-induced diseases. Specifically, the use of neutralizing antibodies in passive immunotherapies is of central importance to the present invention.
Passive immunization of HIV-1 infected humans using human sera containing polyclonal antibodies immunoreactive with HIV has been reported. See for example, Jackson et al., Lancet, Sep. 17:647-652, (1988); Karpas et al., Proc. Natl. Acad. Sci., USA, 87:7613-7616 (1990).
Numerous groups have reported the preparation of human monoclonal antibodies that neutralize HIV isolates in vitro. The described antibodies typically have immunospecificities for epitopes on the HIV glycoprotein gp160 or the related glycoproteins gp120 or gp41. See, for example Karwowska et al., Aids Research and Human Retroviruses, 8:1099-1106 (1992); Takeda et al., J. Clin. Invest., 89:1952-1957 (1992); Tilley et al., Aids Research and Human Retroviruses, 8:461-467 (1992); Laman et al., J. Virol., 66:1823-1831 (1992); Thali et al., J. Virol., 65:6188-6193 (1991); Ho et al., Proc. Natl. Acad. Sci., USA, 88:8949-8952 (1991); D""Souza et al., AIDS, 5:1061-1070 (1991); Tilley et al., Res. Virol., 142:247-259 (1991); Broliden et al., Immunol., 73:371-376 (1991); Matour et al., J. Immunol., 146:4325-4332 (1991); and Gorny et al., Proc. Natl. Acad. Sci., USA, 88:3238-3242 (1991). For a current review of pathogenesis of HIV infection and therapeutic modalities including the use of passive immunity with anti-HIV antibodies, see Levy, Microbiol. Rev., 57:183-289 (1993).
To date, none of the reported human monoclonal antibodies have been shown to be effective in passive immunization therapies. Further, as monoclonal antibodies, they all each react with an individual epitope on the HIV envelope surface glycoproteins, gp120 or gp160, or against the V3 loop of gp120 or against the envelope transmembrane glycoprotein, gp41. The epitope against which an effective neutralizing antibody immunoreacts has not been identified.
There continues to be a need to develop human monoclonal antibody preparations with significant HIV neutralization activity. In addition, there is a need for monoclonal antibodies immunoreactive with additional and diverse neutralizing epitopes on HIV gp120. Additional (new) epitope specificities are required because, upon passive immunization, the administered patient can produce an immune response against the administered antibody, thereby inactivating the particular therapeutic antibody.
Furthermore, the well documented ability of HIV to mutate its envelope glycoprotein structure and thereby alter its reactivity with the immune system of an infected host produces variant xe2x80x9cfield isolatesxe2x80x9d which compromise the utility of individual antibody preparations immunoreactive with an individual laboratory strain of HIV. Existing antibody preparations tend to be less potent against primary field isolates of HIV than against laboratory strains. Moore et al., Perspectives in Drug Discovery and Design, 1:235-250 (1993). In addition, no reported human monoclonal antibody has been shown to be effective at neutralizing multiple strains of HIV. Therefore, there also continues to be a need for a human monoclonal antibody with the ability to neutralize multiple different strains of HIV.
2. Human Monoclonal Antibodies Produced from Combinatorial Phagemid Libraries
The use of filamentous phage display vectors, referred to as phagemids, has been repeatedly shown to allow the efficient preparation of large libraries of monoclonal antibodies having diverse and novel immunospecificities. The technology uses a filamentous phage coat protein membrane anchor domain as a means for linking gene-product and gene during the assembly stage of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial libraries. Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991). Combinatorial libraries of antibodies have been produced using both the cpVIII membrane anchor (Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366, 1991) and the cpIII membrane anchor. Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991).
The diversity of a filamentous phage-based combinatorial antibody library can be increased by shuffling of the heavy and light chain genes (Kang et al., Proc. Natl. Acad. Sci. USA, 88:11120-11123, 1991), by altering the CDR3 regions of the cloned heavy chain genes of the library (Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461, 1992), and by introducing random mutations into the library by error-prone polymerase chain reactions (PCR). Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992).
Filamentous phage display vectors have also been utilized to produce human monoclonal antibodies immunoreactive with hepatitis B virus (HBV) or HIV antigens. See, for example Zebedee et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992); and Burton et al., Proc. Natl. Acad. Sci., USA, 88:10134-10137 (1991), respectively. Human monoclonal antibodies displayed on the surface of bacteriophage through the use of phage vectors, where the antibodies are specific for HIV-1 antigens, gp120 and gp41, have been generated through screening of combinatorial libraries. The resultant antibodies have been shown to be immunoreactive with HIV and to neutralize HIV. See, Barbas et al., J. Mol. Biol., 230:812-823 (1993); Williamson et al., Proc. Natl. Acad. Sci., USA, 90:4141-4145 (1993); Burton et al., Chem. Immunol., 56:112-126 (1993); and Barbas et al., Proc. Natl. Acad. Sci., USA, 89:9339-9343 (1992).
While the above-described phage display-derived anti-HIV antibodies have been shown to neutralize HIV infection, the screened antibodies are representative of the immune repertoire of an immunized or infected host. However, the heavy and light chain pairings isolated for their affinity for an antigen in vitro are not necessarily paired in vivo. Although the phage display system allows for unique pairing of heavy and light chains, in many cases affinity selection restores the approximate pairings. Burton et al., Nature, 359:782-783 (1992). While such immunized sources or immune priming by natural infection provides useful antibody libraries for some antigens, it is not always possible to acquire such libraries.
Although anti-HIV-1 neutralizing antibodies have been obtained through screening of phage libraries prepared from HIV-1 positive donors, the resultant antibodies are limited in specificity and affinity by the heavy and light chain amino acid residue sequences.
The diversity of a filamentous phage-based combinatorial antibody library, however, can be increased by shuffling of the heavy and light chain genes obtained from an initial screen of a library (Kang et al., Proc. Natl. Acad. Sci., USA, 88:11120-11123, 1991). Another approach is to introduce random mutations into the heavy and light chain genes by error-prone polymerase chain reactions (PCR). Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580, 1992). Mutagenesis of proteins has been utilized to alter the function, and in some cases the binding specificity, of a protein. Typically, the mutagenesis is site-directed, and therefore laborious depending on the systematic choice of mutation to induce in the protein. See, for example Corey et al., J. Amer. Chem. Soc., 114:1784-1790 (1992), in which rat trypsins were modified by site-directed mutagenesis. More recently, Riechmann et al., Biochem., 32:8848-8855 (1993), described the use of site-directed mutagenesis and phage display techniques prior to screening the randomized library to increase the affinity of a single-chain Fab fragment specific for the hapten 2-phenyloxazol-5-one.
A preferred approach, in order to more extensively sample the potential of antibody structure and function, is the preparation of semisynthetic antibodies in the context of phage display. In these molecules, one or more of the complementarity determining regions (CDR) of the cloned heavy or light chain genes obtained from screening of the library are altered resulting in new variable domain amino acid residue sequences. Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992). Unlike antibodies cloned from a particular donor, semisynthetic antibodies can have CDR of any size with any sequence, thereby increasing the potential to obtain antibodies having new specificities and affinities.
Synthetic Fab heterodimers specific for HIV-1 glycoproteins having enhanced affinity, specificity and neutralizing capacities as compared to the previously characterized antibodies have now been discovered. The new synthetic HIV-1-specific Fab heterodimers are obtained through the use of the synthetic method of randomly mutagenizing the complementarity determining regions (CDR) of the heavy and light chain genes encoding a recombinant Fab antibody to produce an antibody that binds to and neutralizes HIV.
The randomly mutagenized neutralizing antibodies define new epitopes on HIV, particularly on HIV glycoprotein gp120, thereby increasing the availability of new immunotherapeutic human monoclonal antibodies that exhibit higher affinity binding to the epitope as compared to antibodies selected from a nonrandomized combinatorial library.
The invention provides synthetic human monoclonal antibodies that neutralize HIV more efficiently than antibodies selected from non-randomized combinatorial libraries. Also provided are amino acid sequences which confer the enhanced neutralization function to the antigen binding domain of a monoclonal antibody, and which can be used immunogenically to identify other antibodies that specifically bind and neutralize HIV. The synthetic monoclonal antibodies of the invention find particular utility as reagents for the diagnosis and immunotherapy of HIV-induced disease.
A major advantage of the monoclonal antibodies of the invention derives from the fact that they are encoded by a human polynucleotide sequence, i.e., they are human antibodies. Thus, in vivo use of the monoclonal antibodies of the invention for diagnosis and immunotherapy of HIV-induced disease greatly reduces the problems of significant host immune response to the passively administered antibodies which is a problem commonly encountered when monoclonal antibodies of xenogeneic or chimeric derivation are utilized.
Another major advantage of the human monoclonal antibodies of the present invention is that the antibodies have dramatically increased immunoaffinity for the target antigen, making the antibodies particularly potent both diagnostically and therapeutically.
In one embodiment, the invention contemplates a synthetic human monoclonal antibody capable of immunoreacting with and neutralizing human immunodeficiency virus (HIV). A synthetic monoclonal antibody has the capacity to reduce HIV infectivity titer in an in vitro virus infectivity assay by 50% at a concentration of less than 100 nanograms (ng) of antibody per milliliter (ml) of culture medium in the assay. In preferred embodiments, the monoclonal antibody reduces infectivity titers 50% at a concentration is less than 20 ng/ml, and preferably less than 10 ng/ml.
A preferred synthetic monoclonal antibody is a Fab fragment. More preferred are synthetic monoclonal antibody molecules that immunoreact with an HIV glycoprotein, particularly the HIV glycoprotein gp120.
The invention also describes human monoclonal antibodies, and their method of preparation, which exhibit enhanced or improved virus neutralization capacity for multiple different strains of HIV, i.e., increased breadth of virus strain neutralizing capacity. Thus, a preferred human monoclonal antibody has the ability to neutralize a preselected first HIV strain as described above, and further has the capacity to reduce the HIV infectivity titer of a second field strain of HIV in the in vitro virus infectivity assay by 50% at a concentration of less than 10 micrograms (ug) of antibody per milliliter (ml). By neutralizing multiple strains of HIV, the present antibodies exhibit strain crossreactivity and multi-strain neutralizing abilities.
A preferred synthetic human monoclonal antibody has the binding specificity of a monoclonal antibody comprising a heavy chain immunoglobulin variable region amino acid residue sequence selected from the group consisting of SEQ ID NOs 2, 3, 4 and 5. Another preferred synthetic human monoclonal antibody has the binding specificity of a monoclonal antibody comprising a light chain immunoglobulin variable region amino acid residue sequence in SEQ ID NO 6.
In preferred embodiment, the invention describes the neutralizing antibodies as being immunoreactive with HIV glycoprotein gp120 with a dissociation constant (Kd) of about 1xc3x9710xe2x88x928 M or less, preferably from about 1xc3x9710xe2x88x929 M to about 1xc3x9710xe2x88x9230 M, more preferably from about 1xc3x9710xe2x88x9210 M to about 1xc3x9710xe2x88x9211 M, and most preferably from about 1xc3x9710xe2x88x9211 M to about 1xc3x9710xe2x88x9212 M.
A preferred human monoclonal antibody having the above high-affinity binding specificity comprises a heavy chain immunoglobulin variable region amino acid residue sequence selected from the group consisting of SEQ ID NOs 1, 2, 3, 4, 5, 54, 55, 56, 57, 58, 59, 89, 90, 91 and 92, and conservative substitutions thereof. In addition, a preferred human monoclonal antibody having the above high-affinity binding specificity comprises a light chain immunoglobulin variable region amino acid residue sequence selected from the group consisting of SEQ ID NOs 6, 69, 70, 73, 75, 76, 77, 79, 80, 82, 83, 84, 85, 86, 87 and 88, and conservative substitutions thereof. Particularly preferred is a human monoclonal antibody wherein the monoclonal antibody has the binding specificity of a monoclonal antibody having heavy and light chain immunoglobulin variable region amino acid residue sequences in pairs selected from the group consisting of SEQ ID NOs 2:6, 3:6, 4:6, 5:6, 3:69, 3:70, 3:73, 3:75, 3:76, 3:77, 3:79, 3:80, 3:82, 3:83, 3:84, 3:85, 3:86, 3:87, 54:6, 55:6, 56:6, 57:6, 58:6, 59:6, 89:6, 89:88, 90:86, 90:88, 91:6, 91:88 and 92:88, and conservative substitutions thereof.
Also contemplated are methods of producing a synthetic anti-HIV monoclonal antibody using random mutagenesis methods for sequentially mutagenizing one or more preselected domains of the immunoglobulin heavy chain, preferably a complementarity determining region (CDR), and subsequently selecting for antibodies which strongly immunoreact with and neutralize HIV.
In another embodiment, the invention describes a polynucleotide sequence encoding a heavy or light chain immunoglobulin variable region amino acid residue sequence portion of a synthetic human monoclonal antibody of this invention. Also contemplated are DNA expression vectors containing the polynucleotide, and host cells containing the vectors and polynucleotides of the invention.
The invention also contemplates a method of detecting human immunodeficiency virus (HIV) comprising contacting a sample suspected of containing HIV with a diagnostically effective amount of the synthetic monoclonal antibody of this invention, and determining whether the synthetic monoclonal antibody immunoreacts with the sample. The method can be practiced in vitro or in vivo, and may include a variety of methods for determining the presence of an immunoreaction product.
In another embodiment, the invention describes a method for providing passive immunotherapy to human immunodeficiency virus (HIV) disease in a human, comprising administering to the human an immunotherapeutically effective amount of the synthetic monoclonal antibody of this invention. The administration can be provided prophylactically, and by a parenteral administration. Pharmaceutical compositions containing one or more of the different synthetic human monoclonal antibodies are described for use in the therapeutic methods of the invention.